Investigating microRNA-target interactions during skeletal muscle development in chicken embryos

Viaut, Camille (2017) Investigating microRNA-target interactions during skeletal muscle development in chicken embryos. Doctoral thesis, University of East Anglia.

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MicroRNAs (miRNAs), short non-coding RNAs, which act post-transcriptionally to regulate gene expression, are of widespread significance and have been implicated in many biological processes during development and disease, including muscle disease. In addition to the myomiRs, which are miRNAs highly enriched in striated muscles, recent advances in sequencing technology and bioinformatics led to the identification of a large number of miRNAs in vertebrates and other species. However, for many of these miRNAs specific roles, in particular during myogenesis, have not yet been determined.
Here, I investigated the potential functions of miR-128, confirmed an interaction with one of its candidate targets, Eya4, and looked at the impact of its knock-down on skeletal myogenesis in the chicken embryo.
The expression pattern of miR-128, as well as 22 other somitic miRNAs, were characterised by LNA in situ hybridisation (LNA ISH).
Eya4 was identified as a candidate ‘muscle’ target of miR-128 by computational analysis. Its expression pattern was characterised; miR-128 and Gga-Eya4 displayed similar profiles in developing somites. Using the miRanda algorithm potential miRNA binding sites were identified in the 3’ untranslated region (UTR) of other transcription factors, which along with Eya4 are members of the PAX-SIX-EYA-DACH (PSED) network (Six1/4, Eya1/2/3, and Dach1).
These miRNA/target interactions were examined in vitro and in vivo. Gga-Eya4 was confirmed as a target of miR-128 as well as miR-206 by luciferase reporter assays. MiR-128/Gga-Eya4 interaction was validated by RNA ISH and RT-qPCR after antagomiR (AM)-128 injection in chicken embryos. Knock-down of miR-128 resulted in a significant de-repression of Gga-Eya4 expression; an increase in Gga-Six4 and Gga-Pax3 expression was also observed, whereas Gga-MyoD1 expression was decreased.
With this project, using a combination of cell-based experiments and animal studies, I showed that miR-128 could play an important role in the regulation of skeletal myogenesis in the chicken embryo by targeting Gga-Eya4, a member of the PSED network.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 4971 not found.
Date Deposited: 06 Jun 2017 13:33
Last Modified: 01 Dec 2018 01:38


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